Sterility test is essential to ensure the safe use of sterile products as well as an important link to determine the production cycles of the sterile products. For example, in the pharmaceutical field, strict requirements have been formulated on the sterility tests for injections in the pharmacopeias in various countries. Such requirements essentially form the internationally consistent inspection standards and operation procedures, which have effectively enhanced the sterility guarantee levels of the preparations.
However, the current sterility test methods have certain limitations. First, the sterility test cycles are relatively long, limiting improvements in productivity of the enterprises. It is a common provision in pharmacopeias in various countries that the cultivation cycle of the sterility test shall be 14 days. If it is still impossible to judge the results, additional 7 days are required for subculture. If the result of “false positive” is detected, the tested shall be repeated. This may further extend the ex-factory waiting time and the production cycle. Secondly, the results of sterility test in current pharmacopeias are mainly made by visual observation of the turbidity of culture medium caused by the massive development of the microorganism, which is greatly influenced by the operation experience of the observers. The automation level is low, and there exists subjectivity to some extent. In addition, there are still risks to determine the sterilization conditions of the sample by the turbidity of the culture medium simply by visual operations: Turbidity not related to the growth of microorganism is difficult to be excluded in visual observation; and it is even more difficult to identify slowly developing microbial contamination that has not caused the turbidity of the culture medium within the specified inspection time. This may result in judgment of false positive or negative and affect the accuracy and reliability of the results.
In view of the above problems, it has been a focus of researches on sterile preparations domestically and abroad to establish a method for fast identification of the microbial contamination of the sterile preparation, to enhance the sensitivity and accuracy of the inspection, to shorten the inspection time, to enhance the automation level of the inspection and to supplement or replace the current methods. New methods such as microorganism laser light scattering method, bioluminescence inspection method, and PCR amplification inspection method have been established. These new methods have enhanced our ability to inspect microbial contaminations. However, the application of such methods are still restricted by factors such as the particle sizes of the microorganisms, interference from other particles, complicated operations, expensive instruments and preparations, or lack of universality of the methods (limited to certain types of microorganisms with narrow applications). Therefore, new inspection methods based on the life cycles and growth characteristics of microorganisms are needed.
According to theories of biothermodynamics, all biological activities are accompanied by metabolisms and transformations of energy and substance. Such energy may be monitored with a microcalorimetry system. A microcalorimetry system is a sensitive, fast, convenient, multichannel and real-time online monitoring instrument system. In recent years, the inventors' group has been using microcalorimetric methods to inspect the thermal effects during the growth of microorganisms for quality control and efficacy evaluation of medicines. Experience and results have been achieved to some extent. According to the studies, under proper conditions, the growth of a microorganism exhibits certain patterns and characteristics. Therefore, a new sterility test method may be established based on the microcalorimetric method.
Principles of this invention: Based on the functions available for the microcalorimeter to detect the thermal effects during the growth of the microorganism, transcribe the fingerprint characteristic thermograms for microorganisms of different survival conditions and different types in the microcalorimeter, and establish a standard archive for data analysis. Then transcribe the thermograms of the samples to be tested. On condition that the sterilization of the samples are not at all or not thoroughly made and is contaminated by microorganism, a trend of growth of microorganism may be represented in the thermogram for the sample. Compared with the standard archive established to quickly select the contaminated samples and preliminary determine the types of microorganisms that have contaminated the samples
General operations of the microcalorimeter: Place the microorganism strain into the microcalorimetric ampoule for specific culture medium; then place the ampoule into the microcalorimeter detection channel; record the variations of the heat generated by the growth of the microorganism. However, when making sterile inspection with microcalorimeter, there exists a major defect in the operation links, which is, when conducting the sterility test, as the ampoule structure to be used together with the microcalorimeter is impossible to be sealed during the injection of the samples and culture mediums, which cannot meet the requirements in the microbial contamination inspection (sterility test) for the products of isolation with external environments (to avoid secondary contamination), enrichment of microorganisms and elimination of the antibacterial activity of the products. This may also cause the samples to be contaminated by external factors, leading to a judgment of false positive. Therefore, improvements are required on the ampoule of the microcalorimeter when making sterility test by the microcalorimetric method. Design guidelines for the fully-enclosed bacteria collecting ampoule incubator under this invention: (1) sterility: Apply suitable sterilization method to ensure the sterility of the bacteria collector; (2) tightness ensure effective isolation of the internal part of the system with external requirement (3) bacteria collection: equip necessary bacteria collection device for enrichment of microorganisms and elimination of the antibacterial activity of the products, equip suitable filter membranes according to the features of the samples to be tested; (4) thermal sensitivity: the system may enable sensitively detection of the heat generated by the growth and metabolism of the microorganism by the calorimeter; (5) pressure resistance: the system is able to meet the negative pressure requirements during the process of bacteria collection without damaging the microorganisms; (6) tolerance: the sample should be able to meet the requirements on sterility test on samples and be provided with sufficient inspection capacities; (7) simplicity: the system should be provided with convenient operations, high automation performance and functions of automatic result indication; (8) economy: the system should be economic and easily accessible and should be easy for batch production and generalization.